The regulation of virulence factor gene expression plays a central role in the pathogenesis process. Members of the Mycobacterium fortuitum complex provide an excellent model system for identification and study of virulence factor genes that can play a role in pathogenesis of important human mycobacterial diseases. These bacteria are fast- growing, pathogenic for cultured macrophages and animals, and only rarely pathogenic for humans; they are therefore more amenable to experimental manipulation than slow-growing ones, including ones that are more strictly human pathogens. Specific aims are: (i) to develop a mariner-based transposon mutagenesis method for isolation of M. fortuitum mutants; (ii) to isolate transposon-induced M. fortuitum mutants incapable of in cellulo growth (Icg) within cultured macrophages; and (iii) to isolate in vivo growth defective (Ivg-) mutants using an animal (mouse or goldfish) model system. Continued studies of these mutants are expected to form the basis of a long term research project aimed towards understanding the role of the respective virulence factor (icg or ivg) gene products in disease and how to combat the infectious process by interfering with their mode of action. Results should also provide new insights into pathogenesis mechanisms for human pathogenic mycobacterial species and the design of new antibacterial agents to defend against them.